One-step reduction and alkylation of proteins

ABSTRACT

The present invention relates generally to the field of analyzing proteins and more specifically to the reduction and alkylation of protein samples during protein analysis. A novel composition and method for reducing and alkylating proteins is disclosed. A novel reagent including a combination of a volatile reducing agent, a volatile alkylating agent, and a volatile solvent is used for a one-step reduction and alkylation of proteins allowing the protein sample to remain in the same container during the reduction and alkylation processes.

RELATED APPLICATIONS

This application is a continuation-in-part application of U.S.application Ser. No. 10/654,062.

FIELD OF THE INVENTION

The invention relates generally to the field of analyzing proteins andmore specifically to the reduction and alkylation of protein samplesduring protein analysis.

BACKGROUND AND SUMMARY OF THE INVENTION

Many proteins such as lysozyme are formed from folded polypeptides whichare held in place by disulfide bonds that link the folds. Analysis ofproteins often involves restriction or cutting apart of the polypeptidesby enzymes, such as trypsin, at specific locations of the protein. Afterthe protein has been cut apart, specific sites on the protein can bestudied. Sometimes, the specific locations where the enzyme is to cutapart the protein is inaccessible to the enzyme because of the folding.

One method for unfolding the polypeptides is to reduce the disulfidebonds linking the folded polypeptides. Reduction is accomplished byadding a reducing agent to the protein. After the proteins have beenreduced, typically they are alkylated to avoid reforming of thedisulfide bonds. An alkylating agent is added to the protein to alkylateit. The protein is then prepared for restriction or cutting apart of thepolypeptides by enzymes. Usually the reduction step involves incubatingthe protein sample at a specified temperature followed by multiplewashings or desalting steps. After the washings, the alkylation step iscarried out and also usually involves incubation followed by multiplewashings or desalting.

Common prior art reducing agents used to reduce proteins includedithiothrediol (DTT) and tricarboxyethylphosphene, neither of which arevolatile under a partial vacuum at around room temperature. One methodof reducing a protein involves incubating the protein with one of areducing agent followed by three washing steps. After the reducing step,the protein is then incubated with an alkylating agent such asiodoacetamide or iodoacetic acid, after which the sample is then againsubjected to multiple washing steps. Iodoacetamide and iodoacetic acidare also not volatile under a partial vacuum at about room temperature.Although the method for reducing and alkylating proteins described aboveis common, it is tedious, time consuming, and provides multipleopportunities to lose all or a portion of the protein sample, especiallywhen small samples are involved. The present invention is directed todecrease the amount of time involved in the reduction and alkylation ofprotein samples and reduce the risk of losing all or a portion of theprotein sample.

One embodiment of the present invention includes a compositionconfigured to modify proteins, the composition comprising a singlevolatile reagent. Another embodiment of the present invention includes amethod of modifying proteins comprising the steps of providing avolatile reagent configured to reduce and alkylate the proteins, thereagent including a sufficient amount of solvent to substantiallydissolve the proteins, and dissolving the proteins in the volatilereagent. Another embodiment of the present invention includes a kit forone-step reduction and alkylation of a protein sample, the kit includinga volatile reducing agent configured to reduce the protein sample, avolatile alkylating agent configured to alkylate the protein sample, anda volatile solvent configured to substantially dissolve the proteinsample.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a mass spectrum for a sample of lysozyme taken on a MALDI massspectrometer.

FIG. 2 is a mass spectrum of protein samples after 30 minutes, 60minutes, and 120 minutes in incubation at 37° C. in a solution having apH of about 10.

FIG. 3 is a mass spectrum of protein samples after 30 minutes, 60minutes, and 120 minutes in incubation at 37° C. in a solution having apH of about 8.8.

FIG. 4 is a mass spectrum of protein samples after 30 minutes, 60minutes, and 120 minutes in incubation at 37° C. in a solution having apH of about 8.

FIG. 5 is a mass spectrum of protein samples after 30 minutes, 60minutes, and 120 minutes in incubation at room temperature in a solutionhaving a pH of about 10.

FIG. 6 is a mass spectrum of protein samples after 30 minutes, 60minutes, and 120 minutes in incubation at room temperature in a solutionhaving a pH of about 8.8.

FIG. 7 is a mass spectrum of protein samples after 30 minutes, 60minutes, and 120 minutes in incubation at room temperature in a solutionhaving a pH of about 8.

FIG. 8 is a mass spectrum of protein samples taken at time periods of 60minutes and 120 minutes after incubation while varying the concentrationof iodoethanol.

FIG. 9 is a mass spectrum of protein samples taken at time periods of 60minutes and 120 minutes after incubation while varying the concentrationof triethylphosphine.

FIG. 10 is a mass spectrum of protein samples that have been reduced andalkylated using a methylamine reagent and a triethanolamine reagent andsubjected to a 30 minute incubation period.

DETAILED DESCRIPTION

The present invention relates to a composition and method for reducingand alkylating proteins such as lysozyme. The composition to reduce andalkylate the proteins is a reagent including a combination of a reducingagent, an alkylating agent, and a solvent to dissolve the particularprotein. The reducing agent, the alkylating agent, and the solvent areselected from materials that are volatile under a partial vacuum. Thereducing agent, the alkylating agent and the solvent can each be anindividual material or a combination of such materials. For example, itmay be possible for a single composition to reduce, alkylate anddissolve the protein.

The reducing agent may include any reducing agent or combination orreducing agents that are volatile under a partial vacuum. Reducingagents include triethylphosphine and trimethylphosphine or any othersuitable reducing agent that is volatile when subjected to a partialvacuum. Selection of the volatile reducing agent will depend upon itsability to substantially reduce the protein being treated.

The alkylating agent of the composition can include any alkylating agentor combination of alkylating agents that are volatile under a partialvacuum and sufficiently water soluble such that a second phase does notform during the reaction. Iodoethanol and bromoethanol are examples ofsuitable alkylating agents. Any suitable alkylating agent that is watersoluble and also volatile when subjected to a partial vacuum could beused. The selected volatile alkylating agent must be able to adequatelyalkylate the protein sample.

Known compositions for reducing and alkylating proteins have beenunsuitable for use with a solvent that is less than about 75% organic.That is, with known compositions having solvent compositions of greaterthan about 25% water, the reaction mixture undesirably tends to separateinto two phases. In such event, an intervening step of adding smallamounts of organic solvent must be conducted to restore a single phasereaction composition in which alkylation can take place.

Advantageously, the present invention provides volatile compositions forreducing and alkylating proteins in which the protein remainssubstantially dissolved in the composition at solvent compositions fromabout 30% water to about 90% water, more preferably more than about 50%water. The present invention also enables compositions with solventsthat are mostly or entirely aqueous. It has been found that thealkylating reaction can be carried out in solvents that are essentially100% aqueous without precipitating the protein. Adding organic solventto restore a single phase to complete the alkylating reaction isunnecessary because the protein remains substantially dissolvedthroughout the reaction. The present invention provides the advantagethat solvents that are primarily aqueous are suitable for the reaction,whereas for other applications solvents that are mostly organic are alsosuitable.

Suitable alkylating agents include halogenated alcohols. The alkylatingagent should be chosen with a sufficiently small number of carbons tomaintain its volatility and water solubility, both of which decreasewith increasing carbon chain length. Suitable water soluble, volatilealkylating agents include the following halogentated alcohols:iodoethanol, bromoethanol, iodopropanol, bromopropanol, iodomethanol andbromomethanol. Other alkylating agents may include glycols such asethylene glycol and other halogenated alcohols such as fluoroethanol.One of skill in the art would recognize other suitable water solublevolatile alkylating agents for use with the inventive compositionstaught herein.

Generally, the reagent includes a reducing agent, an alkylating agent,and a solvent, all of which are volatile. The solvent is selected todissolve the protein sample. Experimental results have shown that thesolvent may be up to 100% organic solvent. Experiments have also shownthat the solvent may be up to 100% water. In preferred embodiments thesolvent composition is between about 30% to 70% water with the remainderbeing organic solvent. A suitable solvent is acetonitrile, although onehaving skill in the art would understand that other volatile organicsolvents, such as methanol or ethanol, or mixture of solvents could beused. Aqueous mixtures of organic solvents may also be used. Aqueousmixtures of acetonitrile have been used with good results, especially inconnection with dyed proteins held in gels where it was observed toeffectively remove the dye from the immobilized protein.

The one-step method of reducing and alkylating a protein sampleutilizing the reagent first involves the step of selecting suitablevolatile reducing and alkylating agents and a suitable volatile solvent,if needed, to dissolve the protein sample. To create the reagent, theselected reducing agent, alkylating agent, and solvent are combined in asingle container such as a test tube. Next, the protein sample is placedin the container with the reagent after which the reagent and theprotein sample are incubated at a specified temperature for a specifiedtime period to promote the reduction and alkylation processes. Theincubated mixture is then subjected to a partial vacuum to remove theunreacted portion of the reagent, leaving a dried, reduced and alkylatedprotein sample suitable for rehydration and further treatment, such asby enzymes. It should be noted that the components of the reagent couldbe added to the protein sample simultaneously as described above orsequentially. This one-step method of reducing and alkylating a proteinsample can also be used for gas phase reactions.

In an alternative embodiment, the solvent and the reducing agent aremixed together to produce a first mixture before mixing with the proteinsample. The first mixture is then mixed with the protein sample toproduce a second mixture. The alkylating agent is then mixed with thesecond mixture. In another embodiment, the second mixture is incubatedand the solvent and the reducing agent are subsequently removed by heat,vacuum, or any other suitable method after which a mixture containingsolvent and the alkylating agent is added to the dried protein sample.

In the preferred embodiment, the incubation step is carried out at 37°C. for about one hour. Alternatively, the reaction can be conducted atroom temperature (about 23° C.). In the preferred embodiment, thisone-step reduction alkylation process is carried out at elevatedtemperatures to reduce the time of the reaction such that it ispractical for normal laboratory use. The speed of the reduction andalkylation reactions can be increased by buffering the reagent to a highpH, preferably a pH of about 10. The buffer used in the preferredembodiment of the present invention is 0.1M ammonium carbonate, althoughany suitable buffer may be used.

Following the incubation step, the remaining volatile components of thereagent are removed under a vacuum of about 10⁻² mTorr for about anhour, leaving a dried protein material or a dried gel containing theimmobilized protein, depending upon the starting material. The proteincan then be easily resolubulized in any enzyme containing solution forfurther processing.

Any trace amounts of the reducing agent, alkylating agent, or thesolvent that remain in the reduced and alkylated protein sample can beneutralized by the addition of small amounts of a chelated metal, suchas copper (II) citrate, to the material used to resolubulize theprotein. Any other suitable chelated metal may also be used.

The composition and method of the present invention can be used to treatproteins from any common source such as isolated or fractionated proteinsupplied on a gel or, for example, a mixture of proteins found in aserum. Although the step of removing the remaining volatile componentsof the reagent by subjecting the protein/reagent solution to a partialvacuum is described in the preferred embodiment, this step is notnecessary. In an alternative embodiment, following the incubation step,the protein/reagent solution can be air dried by simply allowing thesolution to be exposed to room temperature air for a period of one tofour hours to allow the remaining volatile components to evaporateleaving the dried protein material.

In another alternative embodiment, the remaining volatile components canbe removed by heating the protein/reagent solution to evaporate thevolatile components. In yet another alternative embodiment, a stream ofcompressed nitrogen can be applied to the incubated protein/reagentsolution to remove the volatile components. Any suitable method forremoving the volatile components remaining after the protein/reagentsolution has been incubated could be used in the one-stepalkylation/reduction method.

In another embodiment it is possible to dissolve, reduce and alkylateproteins entirely in the gas phase. In one experiment, lysozyme wasalkylated in about six hours in the gas phase.

To determine the effectiveness of the reduction alkylation reagent, aMALDI mass spectrometer was used. A mass spectrum for reduced andunreduced lysozyme is shown in FIG. 1. As indicated by reference numeral10, the mass of unreduced lysozyme is 14,305, the mass of reducedlysozyme is 14,313 and the mass of reduced and alkylated lysozyme is14,665. Each alkylation adds 44 mass units to the mass of reducedlysozyme. Lysozyme contains 8 cysteines.

To determine the effectiveness of the reagent, the percentage oflysozyme that was reduced and alkylated when subjected to the reagentwas determined. To perform this calculation, the height of peak 10 andpeak 12 of the mass spectra shown in FIG. 1 was measured. The peakindicated by reference numeral 10 is the point at which 7 cysteine havebeen alkylyzed and measures 3.4 mm when measured on the correct scale.The peak indicated by reference numeral 12 is the point at which 8cysteine have been alkylyzed and measures 4.9 mm when measured on thecorrect scale. The total peak height is then 8.3 mm. The fraction of thepeak with 7 alkylated cysteines is determined by dividing the height ofthe 7 alkylated cysteine peak (10) by the total peak height as shown byequation (1). The fraction of the peak with 7 alkylated cysteines equals0.41. Referring now to equation (2), the fraction of the peak with 8alkylated cysteines is the height of the 8 alkylated cysteine peak (12)divided by the total peak height and equals 0.59. The average alkylationvalue is determined by multiplying the number of alkylated cysteines bythe fraction of the peak height for the respective alkylated cysteinesas shown in equation (3).Fraction of peak with 7 alkylated cysteine=3.4/8.3=0.41  (1)Fraction of peak with 8 alkylated cysteine=4.9/8.3=0.59  (2)Average alkylation value=7(0.41)+8(0.59)=7.59  (3)% reaction=(7.59/8)*100=94.8%  (4)The average alkylation value is 7.59. The average alkylation value isthen divided by the number of alkylated cysteines in lysozyme (8) todetermine the percent of lysozyme that has been reduced and alkylatedwhen reacted with the reagent as shown in equation (4).

EXAMPLES

The useful ratio of the reducing agent, the alkylating agent, and thesolvent for the reagent were determined by titration. Titration of thetriethylphosphine reducing agent was done at concentrations of 0.1,0.25, 0.5 and 1.0 volume percent. Substantially complete alkylation, asdetermined by MALDI mass spectrometry analysis, was obtained atconcentrations of 0.25 volume percent and higher as will be described inthe following examples.

Example 1

A purified lysozyme protein (having a mass of 14299.9 obtained by MALDIMS) in aqueous solution was adjusted to pH 10 with 0.1 M ammoniumcarbonate. An equal volume of the present reagent comprising 0.5%triethylphosphine, 2% iodoethanol and 97.5% acetonitrile was added tothe protein solution. The protein/reagent mixture was capped andincubated at 37° C. for one hour and then uncapped and subjected to avacuum for about an hour to produce a dried pellet in the container. Thedried pellet was reconstituted in the same container to the originalvolume with a trypsin solution. The reconstituted protein was determinedby MALDI MS to have a mass of 14650.9, a difference of 351, whichcorresponds to the addition of a 44 Da ethanol to each of the eightcysteines in the reduced and alkylated lysozyme.

Example 2

The procedure of Example 1 was repeated with BSA substituted lysozyme.The BSA showed a gain in mass of 1539, which corresponds to a 44 Daalkylation for each of the 35 cysteines in BSA indicating the reductionand alkylation were successful.

Example 3

An unfractionated serum sample was dried under vacuum. The resultingprotein pellet was then redissolved in the same container with a volumeof 8 M urea, and 0.1 M ammonium carbonate (pH 10) equal to the originalvolume of the serum sample in order to denature the proteins andmaintain their solubility. An equal volume of the present reagentcomprising 0.5% triethylphosphine, 2% iodoethanol and 97.5% acetonitrilewas added to the protein solution. The protein/reagent mixture wascapped and incubated at 37° C. for one hour and then uncapped andsubjected to a vacuum for about an hour to produce a dried pellet in thecontainer. The dried pellet was reconstituted in the same container withfive times the original volume of a trypsin solution. The sites ofalkylation were verified by subjecting the reconstituted material toLC/MS/MS analysis following incubation with trypsin. Sequence coverageas high as 90% has been observed by mass spectral analysis followingtrypsin digestion.

Example 4

A portion of an SDS-polyacrylamide gel containing a protein that wasstained with Coomassie blue was placed in a container. A volume of thepresent reagent comprising 0.25% triethylphosphine, 1% iodoethanol,48.75% acetonitrile, and 50% 0.1 M ammonium carbonate (pH 10) was addedto the container holding the gel-encapsulated, stained protein. Thecontainer was capped and incubated at 37° C. for one hour and thenuncapped and the excess reagent was decanted. The gel piece wasdehydrated with a volume of pure acetonitrile equal to the volume of thereagent for five minutes, the acetonitrile was decanted and the gelplaced under vacuum for about one hour. The dried gel containing theprotein was reconstituted with 5 to 10 μl of a 20 μg/mL solution oftrypsin. Following trypsin digestion sequence coverage as high as 60%has been recorded by mass spectral analysis.

Example 5

In some iterations of the above examples, the distinct odor oftriethylphosphine was detected in the dried material that remainedfollowing vacuuming. Trace amounts (<1 mM) of chelated copper (II)citrate was added to the trypsin solutions to neutralize any remainingtriethylphosphine that could potentially break the disulphide bonds inthe trypsin.

Example 6

A sample of serum separated from blood was diluted with urea in order todenature the proteins in the serum and to keep them soluble. To theserum sample was added an equal volume of the present reagent comprising0.5% triethyphosphene, 2% iodoethanol and 97.5% acetonitrile. Thereagent/serum mixture was incubated for one hour at 37° C. and subjectedto a vacuum for about an hour to produce a dried pellet in thecontainer.

The dried pellet was reconstituted in the same container with 5 foldexcess volume of a trypsin solution.

Example 7

A sample of serum was processed as above but without dilution with urea.Similar results were observed.

Example 8

A portion of gel holding a stained protein sample was obtained. It wasincubated for an hour at 37° C. in a solution comprising 1 mL of 100 nMammonium carbonate (pH 10) and 1 mL of a reagent comprising 97.5%acetonitrile, 2% iodoethanol and 0.5% triethylphosphine. The liquid wasdecanted following incubation and placed under vacuum for an hour toproduce a dried pellet.

The pellet was reconstituted in a liquid trypsin solution containingtrace amounts of cooper (less than 1 mM) in citrate.

Example 9

Forty-five microliters of a 1 mg/mL solution of lysozyme in water wasplaced in a tube. 5 μl of 1 M ammonium carbonate solution having a pH of11 was added to the tube. The tube then received 50 μl of thealkylation/reduction cocktail which consisted of 97.5% acetonitrile, 2%iodoethanol and 0.5% triethylphosphine. The resulting solution was 50%acetonitrile, 1% iodoethanol and 0.25% triethylphosphine. The final pHof the solution was checked on a micro-pH meter and was found to beabout 10. The solution was incubated at 37° C. for 30 minutes, 60minutes and 120 minutes. At the end of each incubation period, 10 μl ofthe protein solution was removed and diluted to 100 μl with water andcaptured on a C4 ZIPTIP. The bound protein was then eluted onto a MALDItarget with 1-2 μl of 50% of acetonitrile, 0.05% trifluoroacetic acidafter which 1 μl of a saturated solution of sinnapinic acid in 33%acetonitrile, 0.067% trifluoroacetic acid was added to the protein spot.The spot was then allowed to dry and the MALDI target was then loaded ina Voyager Pro mass spectrometer and the mass spectra was acquired in thelinear mode. The mass spectra for the protein samples removed at theperiods of 30 minutes, 60 minutes and 120 minutes are shown in FIG. 2.

Based on the spectra represented by reference numeral 20 and thecalculation method described above, 91% of the lysozyme was reduced andalkylated after 30 minutes of incubation. Based on the spectrarepresented by reference numeral 22 and the calculation method describedabove, 95% of the lysozyme was reduced and alkylated after 60 minutes ofincubation. Based on the spectra represented by reference numeral 24 andthe calculation method described above, 100% of the lysozyme was reducedand alkylated after 120 minutes of incubation.

Example 10

Forty-five microliters of a 1 mg/mL solution of lysozyme in water wasplaced in a tube. 5 μl of 1 M ammonium bicarbonate was added to thetube. Next, the tube received 50 μl of the alkylation/reduction cocktailwhich consisted of 97.5% acetonitrile, 2% iodoethanol and 0.5%triethylphosphine. The resulting solution was 50% acetonitrile, 1%iodoethanol and 0.25% triethylphosphine. The final pH of the solutionwas checked on a micro-pH meter and found to be about 8.8. The solutionwas incubated at 37° C. for time periods of 30 minutes, 60 minutes and120 minutes. At the end of each incubation period, 10 μl of the proteinsolution was removed and diluted to 100 μl and captured on a C4 ZIPTIP.The bound protein was eluted onto a MALDI target with 1-2 μl of 50%acetonitrile, 0.05% trifluoroacetic acid after which 1 μl of a saturatedsolution of sinnapinic acid in 33% acetonitrile, 0.067% trifluoroaceticacid was added to the protein spot. The spot was allowed to dry and theMALDI target was loaded into a Voyager Pro mass spectrometer and themass spectra acquired in the linear mode. The resulting mass spectra areshown in FIG. 3.

Based upon the calculation method described above and the mass spectrashown by reference numeral 30, 62% of the lysozyme was reduced andalkylated after the 30 minute incubation period. Based upon thecalculation method described above and the mass spectra shown byreference numeral 32, 78% of the lysozyme was reduced and alkylatedafter the 60 minute incubation period. Based upon the calculation methoddescribed above and the mass spectra shown by reference numeral 34, 96%of the lysozyme was reduced and alkylated after the 120 minuteincubation period.

Example 11

Forty-five microliters of a 1 mg/mL solution of lysozyme in water wasplaced in a tube. Next, 5 μl of 1 M ammonium bicarbonate was added tothe tube and the pH of the solution was adjusted by adding small volumesof 10% formic acid until the final pH of the solution was about 8.0. Thefinal pH of the solution was checked on a micro-pH meter. The tube thenreceived 50 μl of the alkylation/reduction cocktail which consisted of97.5% acetonitrile, 2% iodoethanol and 0.5% triethylphosphine. Thesolution was incubated at 37° C. for time periods of 30 minutes, 60minutes and 120 minutes. At the end of each incubation period, 10 μl ofthe protein solution was removed and diluted to 100 μl and captured on aC4 ZIPTIP. The bound protein was eluted onto a MALDI target with 1-2 μlof 50% acetonitrile, 0.05% trifluoroacetic acid after which 1 μl of asaturated solution of sinnapinic acid in 33% acetonitrile, 0.067%trifluoroacetic acid was added to the protein spot. The spot was allowedto dry and the MALDI target was loaded into a Voyager Pro massspectrometer and the mass spectra acquired in the linear mode. Theresulting mass spectra are shown in FIG. 4.

Based upon the calculation method described above and the mass spectraindicated by reference numeral 40, 42% of the lysozyme in the solutionwas reduced and alkylated in the 30 minute incubation period. Based uponthe calculation method described above and the mass spectra indicated byreference numeral 42, 79% of the lysozyme in the solution was reducedand alkylated in the 60 minute incubation period. Based upon thecalculation method described above and the mass spectra indicated byreference numeral 44, 95% of the lysozyme in the solution was reducedand alkylated in the 120 minute incubation period.

Example 12

Forty-five microliters of a 1 mg/mL solution of lysozyme in water wasplaced in a tube. 5 μl of 1 M ammonium carbonate solution having a pH of11 was added to the tube. The tube then received 50 μl of thealkylation/reduction cocktail which consisted of 97.5% acetonitrile, 2%iodoethanol and 0.5% triethylphosphine. The final pH of the solution waschecked on a micro-pH meter and found to be about 10. The solution wasincubated at room temperature (approximately 23° C.) for 120 minutes. Atthe end of the incubation period, 10 μl of the protein solution wasremoved and diluted to 100 μl of water and captured on a C4 ZIPTIP. Thebound protein was eluted onto a MALDI target with 1-2 μl of 50%acetonitrile, 0.05% trifluoroacetic acid after which 1 μl of a saturatedsolution of sinnapinic acid in 33% acetonitrile, 0.067% trifluoroaceticacid was added to the protein spot. The spot was allowed to dry and theMALDI target was loaded into a Voyager Pro mass spectrometer and themass spectra acquired in the linear mode. The resulting mass spectra areshown in FIG. 4.

Based upon the calculation method described above and the mass spectraillustrated by reference numeral 54, 88% of the lysozyme in the proteinsolution was reduced and alkylated in the 120 minute incubation period.

Example 13

Example 13 was identical to Example 12, except that 5 μl of 1 M ammoniumbicarbonate was added to the original lysozyme in water solution ratherthan 5 μl of 1 M ammonium carbonate. The final pH of the solution wasfound to be about 8.8. All incubation periods and testing methods wereidentical. Based upon the calculation method described above and themass spectra indicated by reference numeral 64 as shown on FIG. 6, 60%of the lysozyme in the original protein solution was reduced andalkylated in the 120 minute time period.

Example 14

Example 14 was identical to Example 13, except that the pH of theoriginal solution was adjusted with small volumes of formic acid untilthe final pH of the solution was about 8.0. All incubation periods andtesting methods were identical. The resulting mass spectra are shown inFIG. 7. Based upon the calculation method described above and the massspectra indicated by reference numeral 74, 60% of the lysozyme in theoriginal protein solution was reduced and alkylated in the 120 minuteincubation period.

Example 15

Forty-five microliters of a 1 mg/mL solution of lysozyme in water wasplaced in a tube with 5 μl of 1 M ammonium carbonate at a pH of 11. Thetube then received 50 μl of the alkylation/reduction cocktail whichconsisted of 97.5% acetonitrile, 2% iodoethanol and 0.5%triethylphosphine. The final pH of the solution was checked on amicro-pH meter and was found to be about 10. The solution was thenincubated at 37° C. for time periods of 60 minutes and 120 minutes. Atthe end of each incubation period, 10 μl of the protein solution wasremoved and diluted to 100 μl with water and captured on a C4 ZIPTIP.The bound protein was then eluted onto a MALDI target with 1-2 μl of a50% acetonitrile, 0.05% trifluoroacetic acid after which 1 μl of asaturated solution of sinnapinic acid in 33% acetonitrile, 0.067%trifluoroacetic acid was added to the protein spot. The spot was allowedto dry, then the MALDI target was loaded into a Voyager Pro mass spectrameter and a mass spectra acquired in the linear mode. The resulting massspectra are indicated by reference numerals 82, 84 as shown in FIG. 8.

Based upon the calculation method described above and the mass spectraindicated by reference numeral 90, 100% of the lysozyme in the originalprotein solution was reduced and alkylated during the 60 minuteincubation period. Based upon the calculation method described above andthe mass spectra indicated by reference numeral 92 as shown in FIG. 8,100% of the lysozyme in the original protein solution was reduced andalkylated during the 120 minute incubation period.

Example 16

Example 16 is identical to Example 15, except the concentration ofiodoethanol in the alkylation/reduction cocktail was modified. For thisexample, the concentration of iodoethanol in the 50 μl solution was0.66% iodoethanol. The resulting mass spectra are shown by referencenumerals 86, 88 on FIG. 8.

Based upon the calculation method described above and the mass spectraindicated by reference numeral 86 as shown in FIG. 8, 95% of thelysozyme in the original protein solution was reduced and alkylatedduring the 60 minute incubation period. Based upon the calculationmethod described above and the mass spectra indicated by referencenumeral 88 as shown in FIG. 8, 95.4% of the lysozyme in the originalprotein solution was reduced and alkylated during the 120 minuteincubation period.

Example 17

Example 17 is identical to Example 15, except the concentration ofiodoethanol in the alkylation/reduction cocktail was modified. For thisexample, the concentration of iodoethanol in the 50 μl solution of 0.2%iodoethanol. The resulting mass spectra are shown by reference numerals82, 84 on FIG. 8.

Based upon the calculation method described above and the mass spectraindicated by reference numeral 82 as shown in FIG. 8, 56% of thelysozyme in the original protein solution was reduced and alkylatedduring the 60 minute incubation period. Based upon the calculationmethod described above and the mass spectra indicated by referencenumeral 84 as shown in FIG. 8, 73% of the lysozyme in the originalprotein solution was reduced and alkylated during the 120 minuteincubation period.

Example 18

Forty-five microliters of a 1 mg/mL solution of lysozyme in water wasplaced in a tube with 5 μl of 1 M ammonium carbonate having a pH of 11.The tube then received 50 μl of the alkylation/reduction cocktail whichconsisted 97.5% acetonitrile, 2% iodoethanol and 0.5% triethylphosphine.The final pH of this solution was checked with a micro-pH meter andfound to be about 10. The solution was then incubated at 37° C. for timeperiods of 60 minutes and 120 minutes. At the end of each incubationperiod, 10 μl of the protein solution was removed and diluted to 100 μlwith water and captured on a C4 ZIPTIP. The bound protein was elutedonto a MALDI target with 1-2 μl of a 50% acetonitrile, 0.05%trifluoroacetic acid after which 1 μl of a saturated solution ofsinnapinic acid in 33% acetonitrile, 0.067% trifluoroacetic acid wasadded to the protein spot. The spot was allowed to dry, then the MALDItarget was loaded into a Voyager Pro mass spectra meter and the massspectra acquired in the linear mode. The resulting mass spectra as shownin FIG. 9.

Based on the calculation method described above and the mass spectraindicated by reference numeral 108, 100% of the lysozyme in the originalprotein solution was reduced and alkylated in the 60 minute incubationperiod. Based on the calculation method described above and the massspectra indicated by reference numeral 110, 100% of the lysozyme in theoriginal protein solution was reduced and alkylated in the 120 minuteincubation period.

Example 19

Example 19 is identical to Example 18, except the concentration of thetriethylphosphine in the 50 μl alkylation/reduction cocktail was 0.166%,instead of 0.5% as in Example 18. The resulting mass spectra areindicated by reference numerals 104, 106 as shown in FIG. 9.

Based on the calculation method described above and the mass spectraindicated by reference numeral 104, 96% of the lysozyme in the originalprotein solution was reduced and alkylated in the 60 minute incubationperiod. Based on the calculation method described above and the massspectra indicated by reference numeral 106, 100% of the lysozyme in theoriginal protein solution was reduced and alkylated in the 120 minuteincubation period.

Example 20

Example 20 is identical to Example 18, except the concentration of thetriethylphosphine and the 50 μl alkylation/reduction cocktail was0.055%, instead of 0.5% as in Example 18. The resulting mass spectra areindicated by reference numerals 100, 102 as shown in FIG. 9.

Based on the calculation method described above and the mass spectraindicated by reference numeral 100, 89.8% of the lysozyme in theoriginal protein solution was reduced and alkylated in the 60 minuteincubation period. Based on the calculation method described above andthe mass spectra indicated by reference numeral 102, 96.5% of thelysozyme in the original protein solution was reduced and alkylated inthe 120 minute incubation period.

Example 21

Forty-five microliters of a 1 mg/mL solution of lysozyme in water wasplaced in a tube with 5 μl of 2 M methylamine (in methanol). The tubethen received 50 μl of alkylation/reduction cocktail which consisted of97.5% acetonitrile, 2% iodoethanol and 0.5% triethylphosphine. The finalpH of the solution was checked on a micro-pH meter and found to be about11.8. The solution was incubated at 37° C. for 30 minutes. At the end ofthe incubation period, 10 μl of the protein solution was removed anddiluted to 100 μl with water and captured of a C4 ZIPTIP. The boundprotein was eluted onto a MALDI target with 1-2 μl of a 50%acetonitrile, 0.05% trifluoroacetic acid after which 1 μl of a saturatedsolution of sinnapinic acid in 33% acetonitrile, 0.067% trifluoroaceticacid was added to the protein spot. The spot was allowed to dry, thenthe MALDI target was loaded into a Voyager Pro mass spectra meter andthe mass spectra acquired in the linear mode. The resulting mass spectrais indicated by reference numeral 120 on FIG. 10.

Based upon the calculation method described above and the mass spectraindicated by reference numeral 120 as shown in FIG. 10, 100% of thelysozyme in the original protein solution was reduced and alkylatedduring the 30 minute incubation period.

Example 22

Example 22 is identical to Example 21 except 1 μl of triethanolamine wasused instead of the 2 M methylamine. The final pH of the solution waschecked on a micro-pH meter and found to be about 11.4. The resultingmass spectra is indicated by reference numeral 122 on FIG. 10.

Based upon the calculation method described above and the mass spectraindicated by reference numeral 122 as shown in FIG. 10, about 95% of thelysozyme in the original protein solution was reduced and alkylatedduring the 30 minute incubation period.

The foregoing description of the invention is illustrative only, and isnot intended to limit the scope of the invention to the precise termsset forth. Although the invention has been described in detail withreference to certain illustrative embodiments, variations andmodifications exist within the scope and spirit of the invention asdescribed and defined in the following claims.

1. A composition for one-step reduction and alkylation of proteins,comprising: a substantially volatile reducing agent; a substantiallyvolatile solvent; and a substantially volatile and water solublealkylating agent.
 2. The composition of claim 1, wherein the compositionsubstantially dissolves the protein at solvent compositions havinggreater than about 30% water.
 3. The composition of claim 1, wherein thecomposition substantially dissolves the protein at solvent compositionshaving greater than about 50% water.
 4. The composition of claim 1,wherein the alkylating agent comprises a halogenated alcohol.
 5. Thecomposition of claim 1, wherein the volatile solvent comprisesacetonitrile.
 6. The composition of claim 1, wherein the alkylatingagent is selected from the group consisting of iodoethanol,bromoethanol, iodopropanol, bromopropanol, iodomethanol, bromomethanoland mixtures thereof.
 7. The composition of claim 1, wherein theconcentration of the alkylating agent is between about 0.1% to about1.0% by volume of the composition.
 8. The composition of claim 1,wherein the reducing agent is selected from the group consisting oftriethylphosphine, trimethylphosphine, tripropylphosphine,tributylphosphine and mixtures thereof.
 9. The composition of claim 8,wherein the concentration of the reducing agent is between about 0.025%to about 0.25% by volume of the composition.
 10. The composition ofclaim 1, wherein the solvent includes water and acetonitrile.
 11. Thecomposition of claim 1, wherein the volatile solvent is up to 50% waterby volume, based on the total volume of the composition.
 12. Thecomposition of claim 1, wherein the volatile reagent includes a bufferconfigured to buffer the pH of the composition to about pH
 10. 13. Thecomposition of claim 12, wherein the buffer is 0.1M ammonium carbonate.14. A composition for one-step reduction and alkylation of a proteinsample, comprising: a reducing agent to reduce the protein sample; analkylating agent to alkylate the protein sample, the alkylating agentbeing water soluble; a solvent to substantially dissolve the proteinsample; and wherein the composition is substantially volatile, andfurther wherein the composition dissolves the protein at solventcompositions of greater than about 30% water.
 15. The composition ofclaim 14, wherein the composition dissolves the protein at solventcompositions of greater than about 50% water.
 16. The composition ofclaim 14, wherein the alkylating agent comprises a halogenated alcohol.17. The composition of claim 14, wherein the alkylating agent isselected from the group consisting of iodoethanol, bromoethanol,iodopropanol, bromopropanol, iodomethanol, bromomethanol and mixturesthereof.
 18. The composition of claim 14, wherein the concentration ofthe alkylating agent is between about 0.1% to about 1.0% of thecomposition by volume.
 19. The composition of claim 14, wherein thereducing agent is selected from the group consisting oftriethylphosphine, trimethylphosphine, tripropylphosphine,tributylphosphine and mixtures thereof.
 20. The composition of claim 19,wherein the concentration of the reducing agent is between about 0.025%to about 0.25% by volume of the composition.